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#1

Robert-123

    Robert-123


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Geplaatst op 18 oktober 2008 - 16:01

Voor mijn profielwerkstuk wilde ik cortisolmetingen doen. Ik kan alleen nergens vinden hoe je cortisol meet, zijn daar ingewikkelde en dure apparaten voor nodig of simpele test-strookjes of zo? Verder dan dat het kan door speekselmonsters kom ik eigenlijk niet. Hopelijk kan iemand me dit vertellen... Alvast bedankt! :D

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#2

anusthesist

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Geplaatst op 18 oktober 2008 - 18:36

Dit artikel vond ik:

MEASUREMENT OF SERUM CORTISOL
— Cortisol has been measured in serum by several methods. Although many of them are no longer in routine use, the important assays are mentioned to permit one to interpret the older literature.

Methods of measurement


Porter-Silber chromogens — Serum cortisol was first measured by assay of Porter-Silber chromogens (17,21-dihydroxy-20-ketosteroids, referred to as 17-hydroxycorticosteroids, or 17-OHCS) [1]. This method is no longer used.

Competitive protein-binding assay
— This assay uses competition for binding sites on cortisol-binding globulin (CBG, or transcortin) to quantify cortisol [2,3]. Its advantage is lack of drug interference; its disadvantage is that prednisolone and several endogenous steroids, some of which may be increased in pregnancy, adrenal carcinoma, congenital adrenal hyperplasia, and after administration of adrenal enzyme inhibitors, bind to CBG and falsely elevate serum cortisol values. Interfering steroids can be removed before assay by solvent partition or thin layer chromatography (TLC).

Fluorometric assay — This assay exploits the fluorescence of 4-11-beta, 21-dihydroxy-3,20-ketosteroids (11-hydroxycorticosteroids, or 11-OHCS) in sulfuric acid and alcohol [4]. Cortisol and corticosterone are detected by this assay; potent synthetic glucocorticoids are not. Its advantages are simplicity and relative specificity; its disadvantage is that spironolactone, quinine, quinidine, niacin, and benzoyl alcohol also fluoresce in those solvents and therefore falsely elevate cortisol values.

Radioreceptor assay — This assay uses the type II glucocorticoid cytosol receptor as a cortisol-binding agent [5]. Its advantage is its specificity for bioactive steroids, including synthetic glucocorticoids; its disadvantage is the limited supply and instability of the receptor. As a result, this assay is not widely available.

Radioimmunoassay — Radioimmunoassays for cortisol use polyclonal or monoclonal antibodies that are raised to a cortisol analogue that has been conjugated to a protein carrier. Each antibody is characterized in terms of its affinity and crossreactivity with other endogenous or exogenous steroids found in serum. Antibody, labeled cortisol tracer, and cortisol standard are used to perform the assay. The results are dependent upon the specificity of the antibody used in the assay. Both liquid-phase and solid-phase assays of requisite sensitivity and specificity are widely available in reference laboratories and in kit form. Serum total cortisol is measured.

Other immunoassays — Variations on radioimmunoassays using fluorescent, chemiluminescent, and other labels in place of radioisotopic labels, and two-site antibody designs (one antibody is bound to a solid substrate, the other carries the radioactive or other label, and the steroid forms a bridge between them) have similar sensitivity and specificity and are available for use in automated analyzers. The results, like those of radioimmunoassay, are dependent upon the specificity of the antibody used in the assay.

Structurally based assays — In contrast to antibody-based assays, structurally based assays (HPLC, mass spectrometry) are highly specific for the cortisol molecule; they also can measure synthetic steroids [6]. The development of high-throughput techniques to simultaneously measure multiple samples makes these labor-intense assays feasible for commercial use [7]. This method separates cortisol from other steroids and steroid metabolites; cortisol is then measured fluorometrically or spectrophotometrically [8].

Normal values — The serum cortisol concentration normally reflects that of corticotropin (ACTH) and therefore has circadian rhythmicity (show figure 1 and show figure 2). Normal values vary with the particular assay. The following values are representative of an average radioimmunoassay; those obtained by competitive protein binding assay would be similar, and fluorometric assay results are about 3 ĩg/dL (85 nmol/L) higher [9].

In normal subjects serum cortisol concentrations are higher in the early morning (about 6 AM), ranging from 10 to 20 ĩg/dL (275 to 555 nmol/L). Serum cortisol concentrations range from 3 to 10 ĩg/dL (85 to 275 nmol/L) at 4 PM, and the concentrations are lowest, less than 5 ĩg/dL (140 nmol/L), one hour after the usual time of sleep (show figure 2).

Interpretation — Cortisol secretion is episodic and the normal ranges are broad. A single serum value, if it falls within the normal range, is inconclusive. An individual can have partial pituitary or adrenal insufficiency but maintain plasma ACTH and serum cortisol concentrations within their respective normal ranges. For these reasons, stimulation or suppression testing should be performed when there is doubt. Nevertheless, samples drawn at the appropriate time for the suspected endocrine dysfunction can be very helpful in excluding adrenal hypofunction or hyperfunction.

* Patients with primary or secondary adrenal insufficiency have low early morning serum cortisol concentrations. If the value is greater than 10 ĩg/dL (276 nmol/L), it is unlikely that the patient has clinically important adrenal insufficiency, whereas if it is less than 3 ĩg/dL (83 nmol/L), the probability of adrenal insufficiency is high. (See "Diagnosis of adrenal insufficiency in adults"). Since serum cortisol is often undetectable one hour after the beginning of sleep, measurement at this time does not identify patients with adrenal insufficiency.

* Patients with congenital adrenal hyperplasia may have normal or low serum cortisol values (corresponding to simple virilizing and "late-onset" CYP21A2 deficiency types) in the early morning.

* Most patients with Cushing's syndrome have early morning serum cortisol concentrations within or slightly above the normal range. In contrast, serum cortisol concentrations one hour after sleep are almost always high (greater than 7.5 ĩg/dL [207 nmol]) and are often equal to the early morning values (ie, they have an abnormal or absent circadian rhythm) [10]. (See "Establishing the diagnosis of Cushing's syndrome").

Important caveats — Cortisol secretion normally reflects ACTH secretion. As a result, the same caveats concerning circadian rhythmicity, stress, and glucocorticoid administration also pertain to it, except that recent hydrocortisone (cortisol) or cortisone administration may result in high serum cortisol concentrations. The longer disappearance half-time of cortisol than of ACTH (about 80 versus eight minutes) and the several minute lag in its secretion after ACTH stimulation tend to damp excursions in serum cortisol relative to those of ACTH.

Several other factors must be considered in interpreting serum cortisol results.

CBG — Serum cortisol concentrations do not correlate well with cortisol production rates unless the CBG concentration is accounted for [11]. Hepatic CBG synthesis is increased by estrogens [12-14], and early morning serum total cortisol concentrations of 50 ĩg/dL (1400 nmol/L) or higher are not unusual during pregnancy or high dose oral contraceptive use [15,16]. Cortisol dissociates rapidly from CBG, so that early morning values are usually normal in these women. Insulin and insulin-like growth factor-1 inhibit CBG secretion in vitro, and serum CBG concentrations inversely correlated with indexes of insulin secretion such as fasting serum glucose concentrations and A1C are values [17]. Serum CBG concentration is increased in obese patients who have glucose intolerance. Some individuals have low levels of CBG on a genetic basis.

Hepatic and renal dysfunction
— Even relatively severe hepatic dysfunction has little effect on serum cortisol concentrations [18]. Renal failure also has little effect on them, although retained cortisol metabolites may interfere in some radioimmunoassays [19].

Thyroid hormone — Thyroid hormone regulates the rate of cortisol metabolism, but hypothalamic-pituitary feedback mechanisms are intact and serum cortisol concentrations are within normal limits in patients with hypothyroidism or hyperthyroidism.

Body weight — Body weight has no appreciable effect on serum cortisol concentrations, but severe malnutrition apparently has a greater inhibitory effect on cortisol metabolism than on cortisol production, increasing serum cortisol concentrations slightly [20].

Age — It requires one year or more for infants to establish an adult sleep-wake cycle, entrain their circadian rhythms, and establish an adult pattern of ACTH and cortisol secretion [21]. Except for these changes in infants and the fact that, for the first several days of life, normal infants produce more cortisone than cortisol and have low serum cortisol concentrations [22], age has no effect on serum cortisol concentrations.

Depression — Major depressive disorders, especially severe melancholic depression, can result in cortisol dynamics similar to those of Cushing's disease [23-25]. However, most ambulatory patients with major depression have normal hour-of-sleep serum cortisol concentrations.

Synthetic glucocorticoids — Exogenously administered glucocorticoids can alter serum cortisol values either directly, if they cross-react with an antibody, leading to spurious elevations, or indirectly, if they suppress the hypothalamic-pituitary-adrenal axis, leading to low values. (See "Diagnosis of adrenal insufficiency in adults").

Cross-reactivity depends upon the specificity of the antibody for cortisol. This possibility is evaluated during the development of antibody-based commercial assays and the results are available in the assay kit instructions, or from the company.

In contrast to antibody-based assays, structurally based assays (HPLC, mass spectrometry) are highly specific for the cortisol molecule; they also can measure synthetic steroids [6]. The development of high-throughput techniques to simultaneously measure multiple samples makes these labor-intense assays feasible for commercial use [7]. Such assays are useful to evaluate surreptitious ingestion of synthetic steroids or potential cross-reaction in an antibody-based assay.

Depending upon the dose and duration of exogenous glucocorticoid administration, serum cortisol values may also be suppressed, reflecting secondary adrenal insufficiency. If this is the case, medications should be tapered rather than stopped for testing. (See "Glucocorticoid withdrawal").

Non-glucocorticoid drugs — Several drugs induce hepatic cytochrome P-450 enzymes that metabolize steroids. Barbiturates, phenytoin, rifampin, aminoglutethimide, and mitotane increase the metabolic clearance of steroids and of metyrapone. They have a preferential effect on synthetic 9-fluoro steroids (eg, dexamethasone and fludrocortisone) as compared with natural steroids.

These drugs do not alter serum cortisol concentrations in normal subjects, but they can interfere with dexamethasone suppression and metyrapone stimulation tests and necessitate increased steroid replacement dosage in patients with adrenal insufficiency.

Alcohol abuse — Alcohol abuse sufficient to increase serum hepatic enzyme concentrations, especially gamma-glutamyltransferase, can cause pseudo-Cushing's syndrome and high serum cortisol concentrations [26].

Sepsis — Patients with severe illness and sepsis have reduced CBG and albumin levels that result in lower serum cortisol levels [27,28].

SERUM FREE CORTISOL — The biologically active fraction of cortisol in serum is free cortisol. Although a variety of methods have been developed for measuring serum free cortisol [5,29-32], they are technically demanding and expensive and are not in general use. However, recent reports of decreased total cortisol levels in sepsis and critical illness have led to increased interest in measurement or calculation of free cortisol levels in measurement or calculation of free cortisol levels in these patients [27,28,33]. (See "Evaluation of the response to ACTH in adrenal insufficiency", section on Critical illness).

CORTISOL PRECURSORS — Several biosynthetic precursors of cortisol, including pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, and 11-deoxycortisol, can be measured by radioimmunoassay directly or after solvent partition and/or chromatography [30,34].

Normal values — The normal values for these compounds are as follows:

* Serum 11-deoxycortisol is undetectable in normal subjects by current assays (ie, <1 ĩg/dL or 30 nmol/L at 8 AM).

* The early morning serum 17-hydroxyprogesterone concentration ranges from 60 to 300 ng/dL (1.8 to 19 nmol/L60 to 300 ng/dL) in men, 20 to 100 ng/dL (0.6 to 3 nmol/L) in women during the follicular phase of the menstrual cycle, 50 to 350 ng/dL (1.5 to 10.6 nmol/L) during the luteal phase, and 600 ng/dL (18 nmol/L) and more by the end of pregnancy.

Interpretation — These assays are not commonly used for assessment of hypothalamic-pituitary-adrenal function, but some of them do have specific applications.

* Serum 17-hydroxyprogesterone can be measured before and after administration of cosyntropin (ACTH) in patients expected to have the 21-hydroxylase (P-450c21) deficiency variant of congenital adrenal hyperplasia [35,36]. Return of the early morning serum 17-hydroxypregnenolone or 17-hydroxyprogesterone concentration to normal can be used as an index of the adequacy of treatment in this disorder [37].

* Serum 11-deoxycortisol can be measured in tests of pituitary ACTH secretory reserve using metyrapone [38]. (See "Metyrapone stimulation tests").

* One or more of these cortisol precursors may be increased in the serum of patients with adrenal carcinoma [39].

MEASUREMENT OF SALIVARY CORTISOL CONCENTRATION — Serum free cortisol diffuses freely into saliva. Therefore, measurements of salivary cortisol more accurately reflect serum free cortisol concentrations than do measurements of serum total cortisol. The salivary cortisol concentration is independent of salivary flow rate [40,41].

Assay — Saliva (2.5 mL) is obtained after rinsing the mouth but before brushing the teeth, either by unstimulated flow or after chewing uncoated gum or a cotton tube (Plain Salivette, Sarstedt, Newton, NC), and can be stored at room temperature for many days [42] or frozen for extended periods. The sample is thawed, centrifuged at 1500 x g for 10 min at 4ēC, and 2 mL of the supernatant is added to 10 mL of dichloromethane [43]. The dichloromethane is aspirated and evaporated, and the dried extract is reconstituted in assay buffer and assayed by competitive protein-binding assay [2,44], radioimmunoassay [44,45], or enzyme immunoassay [46]. Radioimmunoassay of unextracted saliva has also been described [47,48].

With the development of high-affinity antisera that react specifically with the D ring of cortisol, sensitivity has been improved, and interference by other steroids has been minimized.

Normal values
— Salivary cortisol concentrations vary diurnally, with concentration of about 5.6 ng/mL (15.4 nmol/L) at 8 to 9 AM and about 1 ng/mL (2.8 nmol/L) at 11 PM [43,45,48] (show table 1). The values in obese men and women are similar [43]. Additional work is needed to evaluate the late night normal range in older patients with medical illness [49].

Interpretation — Morning salivary cortisol concentrations are decreased in adrenal insufficiency, while late evening salivary cortisol concentrations are increased in Cushing's syndrome. Both the competitive protein-binding assay and cortisol radioimmunoassays crossreact with other steroids. The competitive protein-binding assay crossreacts with 17-hydroxyprogesterone and 11-deoxycortisol, for example; as a result, cortisol values may be artifactually increased in patients with congenital adrenal hyperplasia and adrenal carcinoma or after metyrapone administration. Some radioimmunoassays are more specific. Cortisol can be chromatographically separated from other steroids before assay in these situations [43].

More recently, developments of liquid chromatography mass spectrometry methods with less cross-reactivity than antibody-based methods, may yield fewer false positive results when used for the diagnosis of Cushing's syndrome [50].

Measuring salivary cortisol is especially useful in assessing cortisol secretion serially in ambulatory patients, who can collect multiple samples and store them in a refrigerator or freezer or even at room temperature for several days between clinic visits. They are also helpful in the evaluation of patients suspected of having cyclical Cushing's syndrome [45,51-54]. (See "Establishing the diagnosis of Cushing's syndrome").

SUMMARY

Measurement of total, free, and salivary cortisol has been advocated for the assessment of adrenal function. The results are affected by the following factors:

* The assay methodology affects the normal range. Currently available antibody-based assays cross react with non-cortisol steroids and have a higher upper limit of normal than structurally-based assays such as high pressure liquid chromatography.

* Changes in CBG and albumin, the binding proteins for cortisol, affect total serum levels, but not free levels in the serum or saliva. These proteins may be substantially reduced in critically ill patients, so that total cortisol values may not reflect adrenal function. Conversely, estrogen-induced increases in CBG may mask low cortisol production.

In individuals with normal sleep-wake cycles, cortisol values are lowest around bedtime, and peak in the early morning. This physiologic difference has been used for diagnostic purposes:

* Patients with Cushing's syndrome have elevated late night salivary and serum cortisol values.

* Patients with severe adrenal insufficiency may have low early morning serum cortisol concentrations. If the value is greater than 10 ĩg/dL (276 nmol/L), it is unlikely that the patient has clinically important adrenal insufficiency, whereas if it is less than 3 ĩg/dL (83 nmol/L), the probability of adrenal insufficiency is high.
That which can be asserted without evidence can be dismissed without evidence.

#3

Robert-123

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Geplaatst op 18 oktober 2008 - 20:41

Oei, dat is volgens mij toch een beetje te ingewikkeld voor een profielwerkstuk... Moet maar iets anders bedenken :D

Maar bedankt voor je antwoord :P

#4

anusthesist

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Geplaatst op 18 oktober 2008 - 21:07

Kun je gewoon niet het laboratorium van een ziekenhuis opbellen in de buurt? Misschien
zijn zij gewillig om je erbij te helpen.
That which can be asserted without evidence can be dismissed without evidence.

#5

Robert-123

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Geplaatst op 18 oktober 2008 - 21:22

Ja, valt te proberen.. Bedankt!





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